Download PaCaHa inhibits proliferation of human cancer

Turkish Journal of Medical Sciences
Turk J Med Sci
(2016) 46: 872-876
© TÜBİTAK
doi:10.3906/sag-1503-142
http://journals.tubitak.gov.tr/medical/
Research Article
PaCaHa inhibits proliferation of human cancer cells in vitro
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Saliha EKŞİ , Nebahat EJDER , Fatih YILMAZ , Ayşe ERTÜRK , Cemal SANDALLI *
Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdoğan University, Rize, Turkey
2
Department of Chemistry, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University, Rize, Turkey
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Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University, Rize, Turkey
Received: 27.03.2015
Accepted/Published Online: 27.07.2015
Final Version: 19.04.2016
Background/aim: The aim of this study was to investigate the antiproliferative and cytotoxic effects of a newly synthesized molecule
named paracetamol acetohydroxamic acid (PaCaHa) on human neoplastic cell lines.
Materials and methods: A549, CRL 2923, HeLa, and ARPE were treated with various concentrations of PaCaHa and DMSO (vehicle
control). The cytotoxic/cytostatic effects of PaCaHa were determined after a 24-h incubation period and compared to the DMSO control.
The cytotoxic and antiproliferative effects were determined by the trypan blue dye exclusion and MTT methods.
Results: A higher susceptibility to PaCaHA was found in CRL 2923 and HeLa cells, while A549 and ARPE cells were less responsive to
PaCaHa. The percent of cytotoxicity resulting from 400 µg/mL of PaCaHa were >90 for CRL-2923 and HeLa, 68 for A549, and 64 for
ARPE cells. The cytotoxic difference between CRL-2923/HeLa and ARPE/A549 cells was significant (P < 0.05).
Conclusion: PaCaHa showed dose dependent cytotoxic and antiproliferative effects on three distinct human cancer cell lines. The
differential effect of PaCaHa on different cancer cell lines suggests that PaCaHa could have a potential antitumor effect on specific cancer
types. These results support further comprehensive studies on PaCaHa and its derivatives.
Key words: Paracetamol acetohydroxamic acid, cancer, MTT, CRL-2923, ARPE-19, A549, HeLa
1. Introduction
Uncontrolled proliferation of cells, resulting from genetic
and epigenetic changes in DNA repair and apoptosis,
leads to cancer (1,2). Cancer is currently the second
leading cause of death after cardiovascular diseases in the
world. According to the 2008 World Health Organization
(WHO) cancer report, cancer is responsible for 7.6 million
deaths each year. This report also estimated that worldwide
deaths are likely to rise to over 11 million in 2030 (3).
These statistics emphasize the need for new treatment
strategies. Currently, the mortality and morbidity rates of
cancer remain high despite advances in surgical, chemical,
radiation and hormone therapies, with associated adverse
reactions and resistance (4).
All these factors have led scientists to continue to
develop alternative cancer treatment strategies (5). In this
context, a number of synthetic compounds and natural
products were evaluated for their efficacy. Paracetamol
acetohydroxamic acid (PaCaHa) was selected for synthesis
based on its similarity to compounds that inhibit cell
growth (6,7). In the present study, we examined the in vitro
cytotoxic effect of PaCaHa on human cancer cells (HeLa,
*Correspondence: [email protected]
872
CRL-2923, and A549) and human diploid cells (ARPE-19)
by using thiazolyl blue tetrazolium bromide (MTT) and
trypan blue dye exclusion methods.
2. Materials and methods
2.1. Drug
PaCaHa was synthesized and kindly provided by Prof
Dr Fatih Yılmaz (Recep Tayyip Erdoğan University, Rize,
Turkey). The chemical structure of PaCaHa is shown in
Figure 1.
2.2. Cell lines and cell culture
The adenocarcinomic human alveolar basal epithelial
cell line (A549) and human cervical cancer epithelial cell
line (HeLa) were kindly provided by Prof Dr Fikrettin
Şahin (Yeditepe University, İstanbul, Turkey); the human
endometrial adenocarcinoma cell line (CRL 2923) was a
gift from Prof Dr Bedia Ağaçhan Çakmakoğlu (İstanbul
University, İstanbul, Turkey); and the diploid ARPE-19
retinal pigment epithelial cell line was kindly provided by Dr
Muradiye Acar (Turgut Özal University, Ankara, Turkey).
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-
EKŞİ et al. / Turk J Med Sci
H
H3C
N
O
O
O
N
OH
H
Figure 1. The chemical structure of PaCaHa.
2-yl)-2,5-diphenyltetrazolium bromide (MTT), taxol,
D-PBS, and trypan blue (0.4%) were obtained from Sigma.
Cell lines were maintained in RPMI-1640 (Hyclone)
or DMEM (Gibco) with 10% fetal bovine serum (FBS)
(Gibco) and antibiotics (100 µg/mL streptomycin + 100
U/mL penicillin) (Gibco) in T25 flasks at 37 °C in 5% CO2.
Confluent cells were detached using 0.25% trypsin-EDTA
(Gibco) solution for serial passage. Cells were used in
cytotoxicity assays as stated below.
2.3. Morphological studies and viability test
Stock solutions (50 mg/mL) of PaCaHa, made in DMSO
(Sigma), were dissolved in a medium in order to generate
working concentrations. A549 (1 × 104 cells/well) and
ARPE cells (5 × 104 cells/well) were seeded into 24-well
culture plates in 100 µL of growth medium in triplicate.
After overnight incubation, various concentrations of
PaCaHa (400, 200, 100, 50, and 25 µg/mL) or solely of
the corresponding DMSO (max. 1.6%), were added to the
wells. Taxol (5 nM) was used as a positive control. The
cultures were maintained at 37 °C for 24 h. Then the cells
were collected by trypsinization and the viable cells were
counted by trypan blue exclusion with a hemocytometer.
The percentage of viable cells was calculated using the
following formula: [(Total cells counts – Dead cells
counts)/Total cells counts] × 100. Cellular morphology
was examined under an inverted light microscope with a
10× objective (Olympus).
2.4. Cytotoxic studies using the MTT assay
This assay was performed according to a slight
modification of the procedure as reported by Mossmann
(8). It was determined that MTT assay depended on the
mitochondrial enzyme reduction of tetrazolium dye in
determining cell viability. Briefly, 1 × 104 cells/well were
seeded into 96-well microtiter plates in 100 µL of growth
medium in triplicate and allowed to adhere overnight. The
next day, different concentrations of PaCaHa (400, 200,
100, 50, 25, and 12.5 µg/mL) or solely of the corresponding
DMSO were added into the cells. After a 24-h incubation
period, 10 µL of filter sterilized MTT (Sigma) solution
(5 mg/mL in water) was added to each well and the cells
were incubated for additional 4 h. After the medium was
removed, formazan crystals formed in viable cells during
the MTT treatment and these were dissolved by adding
100 µL of DMSO/well. The plates were then further
incubated at 37 °C for another 20 min and the absorbance
was measured at 570 nm using the ELISA microplate
reader (Thermo, Multiskan GO) (8). All experiments were
performed three times in triplicate.
2.5. Statistical analysis
Growth inhibition was calculated in terms of percentage
by the formula: [(absorbance of control well – absorbance
of sample well)/absorbance of control well] × 100. The
results were analyzed using an independent sample t-test
or unpaired t-test. The results were considered significant
if the P value was lower than 0.05.
3. Results
3.1. Morphological effect
The cells used in this study were morphologically plastic
adherent and exhibited a fibroblast appearance. To verify
the effect of PaCaHa on cell morphology, A549, CRL2923, HeLa, and ARPE-19 cells were exposed to different
concentrations. The morphological features of A549, CRL2923, HeLa, and ARPE-19 cells were examined under an
inverted microscope after 24-h incubation with PaCaHa
(400, 200, 100 µg/mL), taxol, or DMSO. We observed
that morphological changes were directly proportional
to PaCaHa concentration, as shown in Figure 2. No
morphological changes were observed in cells treated with
DMSO alone; however, those treated with taxol (5 nM)
and PaCaHa (400 µg/mL) were rounded, detached from
the surface, and in some cases showed small vacuoles in
their cytoplasm (data not shown). In contrast, PaCaHa and
taxol (used at the same concentration) had less of an effect
on the morphology of the diploid ARPE cells (Figure 2).
These data suggested that PaCaHa has potential anticancer
activity on human cancer cells.
3.2. Viability results
To evaluate the viability of A549 and ARPE-19 more closely,
we further tested them against five different concentrations
of PaCaHa and DMSO. After 24-h incubation, the
percentages of viable cells were determined by trypan blue
dye exclusion (Table). The effect of PaCaHa on A549 and
ARPE cells was dose-dependent. At 400 µg/mL, only 16%
of A549 cells remained viable compared with 73.6% of
ARPE-19 cells. Viability increased to 64% for A549 cells
and 94.6% for ARPE-19 cells at 200 µg/mL (Table). We
further observed that 100 µg/mL of PaCaHa resulted in
86% and 96% increase in viability of A549 and ARPE cells,
respectively, indicating a gradual increase in cell viability.
The presented data clearly indicate that PaCaHa has higher
rates of growth inhibition effects on A549 cancer cells than
on the control ARPE-19 cells.
3.3. Cytotoxic effect
In order to investigate the effects of PaCaHa on human
cancer cell lines, PaCaHa, with five different concentrations
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EKŞİ et al. / Turk J Med Sci
Figure 2. Cytotoxic effects of PaCaHa on human cancer and diploid cell lines. Cells were treated with PaCaHa, taxol, or DMSO for
24 h. Morphological changes were imaged by inverted microscopy. Panel A: PaCaHa (400 µg/mL), Panel B: DMSO (0.8%), Panel C:
Taxol (5 nM), Panel D: Media alone.
Table. Antiproliferative effects of PaCaHa on A549 and ARPE-19 cells. Cells were treated with PaCaHa or
DMSO for 24 h. Cell viability was calculated by trypan blue exclusion test.
Viability (%)
A549 cell lines
Concentrations
PaCaHa
DMSO
PaCaHa
DMSO
400 µg/mL
16
89.9
73.6
95.5
200 µg/mL
64
90
94.6
95.7
100 µg/mL
86
92
96
93.3
50 µg/mL
89.6
92.8
94.7
92.8
25 µg/mL
85
94
93.6
98
(between 12.5 and 400 µg/mL), and DMSO (control) alone
were added to CRL-2923, HeLa, A549, and ARPE-19.
Once added, cytotoxicity was performed using MTT assay.
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ARPE cell lines
As shown in Figure 3, we found that PaCaHa was cytotoxic
in a dose-dependent manner and induced significant cell
death in CRL-2923 and HeLA cell lines. It was determined
EKŞİ et al. / Turk J Med Sci
Cell growth inhibition(%)
100
CRL - 2923
80
HeLa
A549
60
ARPE
40
20
0
12.5
25
50
100
200
Concentration of PaCaHa (µg/mL)
400
Figure 3. Cytotoxic effects of PaCaHa on human cancer and
diploid cell lines. Cells were seeded on 96-well culture plates
and treated with PaCaHa, taxol, or DMSO for 24 h. Cell growth
inhibition was determined by MTT assay.
that 400 µg/mL PaCaHa was highly cytotoxic and led to
>90% cell death in CRL-2923 and HeLa cells (P < 0.05).
However, the same concentration of PaCaHa killed only
68% of A549 cancer cells. We also observed that the
cytotoxic effects of PaCaHa at a concentration of 400
and 200 µg/mL on ARPE-19 cells were 64% and 19.5%,
respectively. We further showed that there was a gradual
decrease in cell cytotoxicity as the concentration of
PaCaHa decreased (200, 100, 50, 25, and 12.5 µg/mL)
with all three different cancer cell lines used in this study
(Figure 3).
In terms of cytotoxicity, the ARPE-19 cells were
significantly less sensitive than CRL-2923 and HeLa
cells (P = 0.025 and P = 0.05, respectively) to PaCaHa,
while the difference between ARPE-19 and A549 was not
significant (P = 0.517). These results indicate that PaCaHa
is more effective against cancer cells than normal cells
and has a selective impact on different types of cancer
cells (Figure 3).
4. Discussion
The 2008 World Health Organization (WHO) cancer
report estimated that worldwide deaths are likely to
rise to over 11 million in 2030; therefore, new, safe, and
effective anticancer agents are needed (5). In recent years,
the anticancer effects of synthetic chemicals and natural
extracts of plants have gained prominence (9). In the past
few years, different varieties of compounds with cytotoxic
and antiproliferative effects on different cancers have
been identified (10–12). The most desired feature of an
anticancer agent is for it to specifically target cancer cells
with minimal or no toxicity in normal cells. There has
been a continuous effort in the medical field to suppress
the growth of cancer. The goal of the present study was
to assess the cytotoxicity of a newly synthesized molecule
PaCaHa in human neoplastic cell lines. The cytotoxic effects
of PaCaHa were demonstrated by MTT and trypan blue
exclusion assays. The trypan blue experiment showed that
approximately 16% of A549 cells, with a concentration of
400 µg/mL pf PaCaHa, survived after treatment, whereas
74% of the viable cells in the control group survived
(ARPE) (Table). In the MTT assay, three cancer cell lines
and one normal cell line were used to assess the cytotoxic
potential of PaCaHa. The results showed that PaCaHa had
significant cytotoxic effects on HeLa and CRL-2923 cancer
cells compared with control ARPE cells (P < 0.05) (Figure
3). Interestingly, the observations also revealed the effects
of PaCaHa to be more prominent in HeLa and CRL2923
cells compared with A549 cancer cells (Figure 3). This
selective effect on different cancer cells (HeLa and CRL2923 vs. A549) suggests that this compound has potential
antitumor effects. The specific mechanisms of the PaCaHa
reaction responsible for its cytotoxic and antiproliferative
activity are unknown, but there are similar compounds
that exert similar effects by altering cell proliferation,
cell differentiation, and gene expression (13). Additional
PaCaHa derivatives will be tested to define the structural
basis for its activity and selectivity against cancer cells, as
well as its viability as an antitumor agent.
In conclusion, the overall findings of this study indicate
that PaCaHa (12.5–400 µg/mL) increased cell cytotoxicity
in a dose-dependent manner and caused selective
cytotoxicity in specific cancer cells.
Acknowledgments
This work was supported by Recep Tayyip Erdoğan
University Research Fund Grants (BAP-2014.102.03.04
and BAP-2013.102.03.4). We would like to thank Prof
Dr Osman B Özgümüş and Prof Dr Zihni A Yazıcı for
their scientific support. We would also like to give our
special thanks to Prof Dr Bedia Ağaçhan Çakmakoğlu
for providing CRL-2923 cells, Prof Dr Fikrettin Şahin for
providing HeLa and A549 cells, Assist Prof Dr Muradiye
Acar for providing the ARPE-19 cells, and Assoc Prof Dr
Yıldıray Kalkan for helping us with the statistical analysis.
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